Method and Apparatus for Quantifying Solutions Comprised of Multiple Analytes

ABSTRACT

A multi-analyte sensor system based on hollow core photonic bandgap fiber and Raman anti-Stokes spectroscopy. The system includes:
         i) an inlet to introduce an analyte sample into an analyzer chamber which analyzer includes;   ii) a measurement system to derive the anti-Stokes spectral peaks and/or spectra of the sample;   iii) a set of reference calibrants corresponding to the analytes of which the sample is primarily comprised;   iv) a second inlet to introduce said calibrants into the analyzer chamber;   v) a second measurement system to derive the anti-Stokes spectral peaks and/or spectra of the calibrants   vi) an outlet through which the sample and calibrants are expelled from the analyzer chamber.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/121,734, filed Sep. 5, 2018, now U.S. Pat. No. 10,768,050, which is acontinuation of U.S. application Ser. No. 13/506,585, filed Apr. 30,2012, now U.S. Pat. No. 10,101,209, which are incorporated herein byspecific reference.

BACKGROUND OF THE INVENTION

The search for an accurate, at-line real time multi-analyte analyzer hasbeen ongoing for many years. The application space for such an analyzeris both diverse and broad, spanning many fields including but notlimited to food and beverage manufacture, medical diagnostics, chemicalanalysis, energy, and especially bioprocessing and biotechnology. Whilethe description of the present invention focuses mainly on biotechnologybased applications, the invention described and claimed, and theprinciples discussed in respect thereto are general in applicability andthe conclusions are not limited to the biotechnology application arena.

The demand for this type of analyzer instrument in many fields is basedon the need to quantitatively analyze multiple analyte species presentwithin a sample in a closed container. Numerous attempts have been madeto apply a wide variety of technologies for this purpose. Whileinstruments exist today that invasively take samples which are thenanalyzed using standard chemical assays in a more or less automatedformat, the preferred analytical solution would be non-invasive andtherefore not require a breach in the wall of the sample container. Someof the most often applied technologies to achieve sample analysis areso-called “label free” optical technologies that probe the system undertest. These techniques are minimally invasive although not actuallynon-invasive and hold the promise of not compromising either the testsample or the system under test.

Linear scatter and absorption based optical analytical techniques haveto date proved to be of only limited use, as they either lackspecificity or require some tag or label to be introduced into thesample, thereby contaminating it and making it unavailable for furtheruse. Early attempts using optics often employed near-infrared (NIR)absorption to measure spectral signatures in order to uniquely identifyand quantify the analytes of interest (see Simultaneous Measurement ofGlucose and in Insect Cell Media Culture by Near Infrared Spectroscopy,Riley et al, Biotechnology and Bioengineering, 55, 1, p. 11, 1997 andDetermination of physiological levels of glucose in an aqueous matrixwith digitally filtered Fourier transform near-infrared spectra, M. A.Arnold and G. W. Small, Anal. Chem. 62, p. 145, 1990). Attempts toquantify biological materials using NIR (near infra-red) spectroscopygenerally fail due to the lack of specificity provided by this type ofspectroscopy. The spectral features found in the NIR are generally notparticularly sharp (narrow) or distinct, thereby making it difficult torecognize the spectral features, and extract individual analyteconcentrations owing to spectral overlap. Specifically, when manyanalytes are present simultaneously, it is difficult if not impossibleeven using sophisticated multi-variate computer-based algorithms toobtain clinically accurate results. Additionally, any system noisecompromises the spectral data as the broad and rolling spectral featuresoverlap and are smeared out. Even determining the exact spectral peaklocations and amplitudes can often be extremely challenging.

More recent spectroscopic investigations have utilized the Ramanscattering effect to interrogate the sample, as Raman spectra aregenerally sharper and more distinct (see Concentration Measurements ofMultiple Analytes in Human Sera by Near-Infrared Laser RamanSpectroscopy, Jianan Y. Qu, Brian C. Wilson, and David Suria, AppliedOptics, 38, 25, p. 5491, 1999 and Rapid, noninvasive concentrationmeasurements of aqueous biological analytes by near-infrared Ramanspectroscopy, Andrew J. Berger, Yang Wang, and Michael S. Feld, AppliedOptics, Vol. 20 35, 1, p. 209, 1996). Raman scattering is a knownnonlinear optical scattering process which involves inelastic scatteringof a photon and an atom or molecule. When light is scattered from anatom or molecule, most photons are elastically scattered such that thescattered photons have the same energy and wavelength (frequency) as theincident photons (i.e., Rayleigh scattering). However, a small fractionof the scattered photons have a frequency different from the frequency25 of the incident photons. This new frequency can be higher and/orlower, but the scattering to the lower energy level occurs with a farhigher probability per unit time. For this inelastic scattering processto proceed, the energy (and momentum) difference is taken up by the atomor molecule in a process whereby an electronic, vibrational, orrotational quantum is excited and/or de-excited.

The inelastic scattering process known as the Raman Effect leads to bothlower and higher energy scattered photons, which are referred to asStokes scattering and anti-Stokes scattering, respectively. The RamanEffect is often modeled as the absorption and subsequent reemission of aphoton via an intermediate vibrational state, having a virtual energylevel. If/when this absorption and reemission of light occurs in Ramanscattering there is an energy exchange between the incident photons andthe molecules. The energy differences are equal to the difference in theelectrical, vibrational or rotational energy-levels of theatom/molecule. In Raman spectroscopy of molecules, the vibrationalenergy shift is the most commonly observed. If a molecule absorbs energyand a lower energy photon is emitted, it is referred to as Stokesscattering. The resulting photons of lower energy have an energydistribution which generates a Stokes spectrum that is “red-shifted”from the incident beam, or equivalently stated, lower in energy than thepump light. If the molecule loses energy (gives up a quantum unit ofenergy from an excited electronic, vibrational, or rotational state) byhaving that energy combine with an incident photon, it is referred to asanti-Stokes scattering. These incident photons are shifted to the higherenergy (blue) side of the incident light.

These differences in energy between incident and scattered photons aremeasured by subtracting the energy of the single-frequency excitationlaser light source from the energy of the inelastically scatteredphotons. The intensities of the Raman bands are dependent on the numberof molecules occupying the different vibrational states, when theprocess began. If the sample is 20 in thermal equilibrium, the relativenumbers of molecules in states of different energy will be given by theBoltzmann distribution:

$\frac{N_{1}}{N_{0}} = {{\frac{g_{1}}{g_{0}}e} - \frac{\Delta E_{v}}{kT}}$

where:

-   -   N₀: number of atoms the lower vibrational state    -   N1: number of atoms in higher vibrational state    -   g₀: degeneracy of the lower vibrational state    -   g₁: degeneracy of the higher vibrations state    -   ΔE_(v): energy difference between these two vibration states    -   k: Boltzmann constant    -   T: temperature Kelvin

It can be seen from the Boltzmann relationship shown above that lowerenergy states will be more highly occupied than the higher energystates. Therefore, the Stokes spectrum will be significantly moreintense than the anti-Stokes spectrum generally by a few orders ofmagnitude. Given that the spontaneous Stokes signal is orders ofmagnitude lower in power than the incident light, the spontaneousanti-Stokes signal in most free space optical systems will be very closein amplitude to the noise flor of the measurement system. Additionally,the scattered light will be Lambertian and therefore the capture of thescattered light for analysis will be limited by a restatement of the2^(nd) law of thermodynamics known as conservation of brightness (seeArt of Radiometry, James Palmer and Barbara Grant, 2010 SPIE, ISBN978-0-8194-7245-8). It is for this reason that the anti-Stokes spectrumhas typically not ben of common or practical use in analyte detectionand identification.

The most commonly employed Raman spectroscopy system for analyteidentification of biological samples is a Raman Stokes system that usesa laser source having a wavelength of 785 or 830 nm. The excitationwavelength can be anywhere from the ultra violet to the infrared, butthe most common wavelengths employed are between 70 nm-90 nm (nearinfrared or NIR) window because laser sources of this wavelength arereadily available and most tissue and biological fluids exhibit minimumabsorption in this wavelength region so 20 that auto-fluorescence (whichresults in a non-uniform baseline shift interference) is reduced. Theseadvantages of the NIR pump region are counter balanced by twolimitations:

-   -   1. The Raman Stokes scattering cross section, (hence signal        intensity) has a 1/λ⁴ dependence, where λ here represents the        wavelength of the excitation source;    -   2. The sensitivity of the most common multi-channel detectors        used in conjunction with 25 NIR optical spectrometers, namely        silicon CCDs (charge coupled devices), falls of rapidly for        wavelengths exceeding 1 pm.

Therefore, in the 785 to 830 nm wavelength regime there is a localoptimum that can be achieved by a tradeoff between Raman cross-section,auto-fluorescence, detector sensitivity, and filter efficiency. Systemsare employed at other wavelengths depending on situation and thewavelength dependence of the Raman cross-section, or if resonance Ramanscattering is utilized (see Achievements in resonance Ramanspectroscopy: Review of a technique with a distinct analytical chemistrypotential, Evtim V. Efremov, Freek Ariese, Cees Gooijer, AnalyticaChemical Acta, Volume 606, Issue 2, 14 Jan. 2008, Pages 19-134). Thisdepends on which part of the atomic or molecular (electronic,rotational, vibrational) spectrum, which is intended to be probed, aswell as the complexity and molecular structure of the analyte speciesand background matrix being measured.

In practice, art workers implementing optical free space spontaneousRaman spectroscopy systems have generally utilized a carefully designedfree space optical system (bulk lenses, reflecting collection systems,or the like) to collect the Raman Stokes scattered light. As 10previously mentioned, given that the Raman scattered light generallyconstitutes what is known as a Lambertian source the detection systemcollection efficiency and coupling to the spectrometer will be limitedby the law of the conservation of brightness (see Art of Radiometry,James Palmer and Barbara Grant, 2010 SPIE, ISBN 978-0-8194-7245-8).

The existing body of scientific literature contains descriptions ofnumerous systems for analyte identification and quantification based onRaman spectroscopy systems. A bio-analysis system was described byBerger et al (Multicomponent Blood Analysis by Near-Infrared RamanSpectroscopy, Andrew J. Berger, Tae-Wong Ko, Irving Itzkan, GaryHorowitzi and Michael S. Feld, Applied Optics, 38, 13, p. 2916, 1999).This particular system was designed to non-invasively measure analytesof medical significance in human blood (e.g.: Glucose, Cholesterol,Triglyceride, Urea, Total Protein, and Albumin). The apparatus utilizeda diode laser emitting at 830 nm, a mirror and lens system to collectthe Raman Stokes light and deliver it to a spectrometer that employed asilicon CD array. This system reportedly yielded quantitative resultsthat approached the accuracy required to be used in clinicalmeasurements. Given that the Raman Stokes scattered light levels werelow and that they were going through a turbid media (skin and blood),Berger at al. used the method of Partial Least Squares (PLS) andtraining sets in order to retrieve the concentration data from thespectral data of multiple analytes simultaneously (see MultivariateCalibration, H. Martens and T. Naes, Wiley, New York, 1989 or Mixtureanalysis of spectral data by multivariate methods, or Windig, W.,Chemom. Intell. Lab. Syst. 4, p. 201-213, 1988). Training sets are theresponse function spectra of the instrument to a known set of analyteswith known concentrations. These training sets are required so that thePLS algorithm can create a set of basis vectors that are then usedcomputationally to determine the concentrations of multiple analytes ina given sample's Raman Stokes spectrum. This sample's total spectrum iscomprised of the spectra of each of the individual analytes hittingaddition of other spectral components or a change in the spectralbaseline), then the results gained using PLS may no longer be correct.This can occur for instance in a biological sample if there is spectralcontent added due to a bacterial infection, or if the sample is modifiedin a way that changes the basis vectors of the training set.Multivariate analysis techniques like PLS are also sensitive to changesin both the signal and/or the training sets, and quantitation errors cancreep in due to noise or other effects that impact either the signalintegrity or the training sets. Also, training sets are in general bothtime-consuming and can sometimes be difficult to create. Finally, ifanything changes the nature of the system by changing the constituentmake up or composition, the training sets may no longer be valid,thereby invalidating the basis vectors and signals. For example, such asituation can easily be envisaged in biological systems where anadventitious agent (bacterial of viral) can change the chemical make-upof a system or in chemical systems where dyes or additives can clearlychange the absorption profiles of the system.

Another complication for this type of system, and indeed for almost alloptical systems including Raman spectroscopy systems used to measurebiological samples, is fluorescence. As both the Raman Stokes signal andauto-fluorescence emissions are red-shifted from the pump light, thereis always overlap between the two signals. This complicates thedetection and identification process as the fluorescence emission oftenobfuscates the Raman Stokes spectrum. A known technique to try andmitigate the effects of auto-fluorescence is to fit it to a high orderpolynomial (typically 5^(th) order) and subtract it out of the spectrum(see Automated Method for Subtraction of Fluorescence from BiologicalRaman Spectra, Lieber and Mahadevan-Jansen, Applied Spectroscopy, 57,11, p. 1363, 2003). While this technique aids in cleaning up thespectrum, it is not obvious that all of the auto-fluorescence isaccurately fitted and that the amplitudes of the spectral features thatare revealed are absolutely or even relatively correct. A publishedexample of this fighting and subtraction technique is shown in FIG. 1(see Quantitative analysis of serum and serum ultrafiltrate by means ofRaman spectroscopy, Rohleder et al, Analyst, 129, p. 906, 2004). Whilethe peaks are more visibly revealed, there is no evidence that theamplitudes of all the features have been maintained in true proportionthereby leading to the potential for quantitation errors when used inconjunction with multi-variate analysis.

Clearly, if the Raman Stokes signal can be increased relative to thefluorescence background, many of these issues can be mitigated. However,the Raman Stokes signal and the fluorescence signal both vary linearlywith the intensity of the pump light, so it is difficult topreferentially generate a Stokes signal with higher relative amplitudeto the fluorescence. The act of subtracting of a generic function asdescribed above can often be the mathematical operation of subtractingsignals of similar magnitude and thereby offers little 15 improvement inthe signal to noise ratio and ads uncertainty to the resulting spectrum.However, in samples that are first passed through a filtration/opticalscattering particle reduction system (e.g., ultrafiltration,centrifugation etc.) the increased Raman Stokes amplitude can result inhigher signal to noise ratios as the filtering reduces theauto-fluorescence and scattering loses (se Quantitative analysis ofserum and serum ultrafiltrate by means of Raman spectroscopy, Rohlederet al., 129, p. 906, Analyst, 2004). Despite these facts, many attemptshave been made to increase the Raman signal including using higher powerpump lasers and increasing the density of the target analyte. Someapproaches have generally utilized clever optical systems topreferentially capture more of the Raman Stokes scattered signal light.

Hollow waveguide technology is one method for increasing the RamanStokes signal that holds promise. The use of hollow core Teflon AF®tubing as an optical waveguide was met with great interest when firstdemonstrated by Altkorn (see Low-loss liquid-core optical fiber for lowrefractive index liquids: fabrication, characterization, and applicationin Raman spectroscopy, Altkorn et al., Applied Optics, 36, 34, p. 892,1997). Teflon AF is one of the 30 few materials with a refractive indexlower (n˜1.29) than many aqueous solutions (n_(water)˜1.33) a propertythat allows it to serve as an optical waveguide. This fact allows manyaqueous solutions to be analyzed using Raman spectroscopy by introducingthe solution into the hollow core Teflon AF waveguide. The increasedconfinement in the core and increased interaction length both act toenhance the Raman signal. Specifically, the Teflon tubing acts as awaveguide as the pump rays introduced into the core and the Raman Stokessignal generated in the core undergo total internal reflection at theliquid/Teflon boundary and are thereby confined primarily within thecore. This results in an increased interaction path and a higher levelof intensity over this path than a comparable free space system.Increases in the sensitivity of the system by a factor of 50 have beenreported, though the general enhancement factor that can be achieved iscorelated to the exact experimental geometry implemented (see IntensityConsiderations in Liquid Core Optical Fiber Raman Spectroscopy, Altkornet al, Applied Spectroscopy, 5, 4, p. 373, 2001 and Raman SensitivityEnhancement for Aqueous Protein Samples Using a Liquid-CoreOptical-Fiber Cell, M. J. Pelletier and Altkorn, Anal. Chem., 73 (6), p1393-1397, 2001).

Unfortunately, small diameter (sub 100 micron inner-diameter) Tefloncapillary tubing is not readily available, and it is therefore difficultto make a single mode waveguide in the near-infrared spectral region.This is because the number of propagating modes in this type ofwaveguide is dependent on the product of the ratio of the core diameterto the wavelength and the square root of the difference between thesquares of the core refractive index and cladding refractive index.Often with the case of Teflon waveguides, the waveguide diameterapproaches a level where it is two orders of magnitude larger than thewavelength of the light propagating so that the mode picture is replacedby a ray optics and numerical aperture description of the lightpropagation within the tube (see Intensity Considerations in Liquid CoreOptical Fiber Raman Spectroscopy, Altkorn et al, Applied Spectroscopy,5, 4, p. 373, 2001). Moreover, Teflon materials can be difficult to workwith as the low surface tension does not allow them to readily bond withother materials. The larger diameter tubing used in the literature hasresulted in waveguides that are not single mode for either NIR pumpwavelengths or the Raman signal. The fact that the waveguide ismulti-spatial mode allows the pump and the Raman Stokes signal to havedifferent spatial profiles and effective velocities in the waveguidethereby limiting the integrated spatial overlap and overall conversionefficiency from the pump to the Raman Stokes signal. Additionally, asTeflon is difficult to bond to, the coupling of the light and fluid intoand out of the fiber is usually accomplished using mechanical fixturing(as opposed to an integrated set of bonded components) which can becumbersome to implement. Finally, there are fundamental limitations tothe density of materials that can be analyzed with Teflon tubing, as therefractive index of the material approaches that of the Teflon cladding.

Another impediment to accurate quantification in the identification ofbiological or other types of samples with optical analyzers based onRaman scattering is the linear optical loss caused by particles in thesample and/or absorption of the pump or the Raman scattered light by thesample. For instance, in whole blood, there is both absorption andscattering in the NIR. The blood cells can create large scattering losesand the Raman Stokes levels can be mediated by direct scattering of theRaman Stokes signal and/or by pump light scattering as well as by pumplight and/or signal absorption. The effect of absorption or scatteringloss 15 can be accounted for if the magnitude of the loss coefficient isknown, but this is often very difficult to determine a priori. Althoughone can devise an instrument to measure the loss coefficients in-situthe overall instrument set-up becomes quite complicated and thereforeimpractical to employ commercially. An example of a laboratory systemwhere this has been implemented is shown in FIG. 3 (see Chemicalconcentration measurement in blood serum and urine samples usingliquid-core optical fiber Raman Spectroscopy, Qi and Berger, AppliedOptics, 46, 10, p. 1726, 2007). Here a Raman Stokes analyzer using aTeflon AF waveguide and simultaneously employed a white lightspectrometer to account for the scattering and absorption in the samplealong the waveguide path. The authors here also reported viable resultsusing this system but required integration times in excess of 10 secondsdespite the waveguide enhancement and the compensation for opticalloses.

Some of the aforementioned issues with Teflon based hollow core fibershave been somewhat overcome with the advent of hollow core photonicband-gap (HCPBG) fibers (see Photonic Crystal Fiber, Philip Russell,Science 17, 29, 5605, p. 358, 2003, U.S. Pat. No. 6,829,421, Hollow CorePhotonic Bandgap Optical Fiber, and Published US Patent Application206/06253, Photonic Crystal Fiber, Method of Manufacturing the CrystalFiber and Method of Connecting the Fiber). The nature of the photonicband-gap allows most gases or liquids to be confined in the core of thefiber, while guiding of the excitation (pump) and scattered (signal)light is supported. Additionally, it allows for the fiber to beconstructed so that the sample of interest is introduced into the corebut the fiber remains single mode or close to single mode for both thepump light and the emitted spectra in the near infrared spectral region.Several groups have implemented Raman Stokes analyzers using HCPBG fiberand others have explored various systems and concepts using this type offiber (se Published US Patent Application 2010/01407, U.S. Pat. No.7,595,82, Stimulated Raman scattering in an ethanol core microstructuredoptical fiber, Yiou et al, Optics Express, 13, 12, p. 4786, 2005, andDetermination of Ethanol Concentration by Raman Spectroscopy inLiquid-Core Microstructured Optical Fiber, Meneghini et al, IEEE SensorsJournal, 8, 7, p. 1250, 2008). U.S. Pat. No. 7,595,82 describes a systemfor identifying homo-nuclear molecules confined to the core of HCPBGfibers. This patent describes the general advantages provided byconfining the radiation and the sample to a hollow core. Published USPatent Application 2010/01407 describes a method for identifyingbiological samples using HCPBG as a Raman biosensor. In addition to ageneral description of how to use the Raman Stokes signal to create abio-analyzer, his patent application discusses how to pick the fibercore diameter or the wavelength of the pump light for a given fiberdesign and a given analyte mixture's index of refraction based on apublished reference. If a commercially available HCPBG fiber comes inspecific discrete core diameters, the single-mode cut-of wavelength isdetermined for a given index of refraction media in the hollow-corebased on the photonic band gap cladding. The above-cited patentapplication focuses solely on Raman Stokes signals as indicated by thespectra shown with a positive wavelength shift and makes no mention ofan anti-Stokes spectrum. We have found that stimulated Raman anti-Stokesbased spectroscopy is also possible with HCPBG fiber. The firststimulated Raman anti-Stokes signal will occur when there is a largeenough population build-up in the first Raman Stokes and Ramananti-Stokes levels to allow for this process.

Recent work was performed by Meneghini et al. (see Determination ofEthanol Concentration by Raman Spectroscopy in Liquid-CoreMicrostructured Optical Fiber, Meneghini et al, IEEE Sensors Journal, 8,7, p. 1250, 2008). Here a HCPBG fiber was used to determine ethanol andsucrose content in a set of samples. In the described system, the HCPBGfiber was spliced to multi-mode fiber on both the input and output endsso that pump light could be coupled in and Raman Stokes light could becoupled out and sent to a spectrometer. The input and output fibers wereattached to the HCPBG fiber using a fusion splicer. The act of fusionsplicing also collapsed the cladding holes around the launch (lightinput) area. Additionally, laser holes were drilled into the side of thefiber allowing the core to be filed with the ethanol and sucrosecontaining solutions that comprised the samples under test. The systemreportedly gave clean and well resolved Raman Stokes spectra, therebyallowing for reasonably accurate quantification of the analyteconcentrations using univariate techniques. Spectral graphs wereobtained with 1 mW and 1 meter of HCPBG fiber as there was nofluorescence to complicate the spectrum and little scattering. Theirtesting, however, did not involve biological samples and/or any sampleshaving large fluorescence and/or scattering/absorptive backgrounds.Additionally, it was clear from their work that very low pump laserpowers (<10 mW) and very short fibers (<2 M) were required to get theirresults.

It is important to note that while all of the previously referencedprior art refers to “Raman scattering”, the term was invariably usedsolely in the context of the Raman Stokes spectrum The only reportedwork known to the present inventors which address identifying analytesusing the Raman anti-Stokes spectrum is described in BiologicalApplications of Anti-Stokes Raman Spectroscopy: Quantitative Analysis ofGlucose in Plasma and Serum by Highly Sensitive Multichannel RamanSpectrometer, Dou et al, Applied Spectroscopy, 50, 10, p. 1301, 1996. Itwas reported by Dou et al, that by employing the complex free spaceoptical collection system they utilized, the anti-Stokes spectrum couldbe used to predict the concentration of glucose in blood serum if theircollection system was implemented. Their optical collection system (asshown in FIG. 3) consisted of a quartz flow cell surrounded by a goldcoated integrating eclipse (it collects or integrates the signal) withtwo holes in it, one small hole 310 that allowed the Argon Ion pumplaser light to pass into the cell, and a small conical shaped hole 320through which the anti-Stokes spectrum escaped and was subsequentlycollimated and sent to a holographic spectrometer and CD array. Theelliptical chamber 315 was designed to optimize the Raman anti-Stokessignal emanating from the quartz flow cell 300. Given the very shortinteraction length of the pump and the analyte and the fact that thespontaneous Raman anti-Stokes signal is emitted into al space, efficientgeneration and collection of the signal is very challenging. Despitetheir carefully designed and implemented free space collection system,they still required 100 mW of pump power at 5145 nm and also needed tointegrate on their CD for 3 seconds to obtain reasonably clear spectra.Also, they simultaneously collected both the Stokes and anti-Stokesspectrum in the presence of heavy fluorescence of the plasma (as shownin FIG. 4). Using the Raman anti-Stokes band at 0130 cm⁻¹ they were ableto create a plot of glucose concentration in blood plasma that matchedthe intensity on the CD with a correlation coefficient of 0.993; as isshown in FIG. 5. They apparently did not require multivariateanalysis/PLS and/or training sets in order to determine theconcentration of the analyte that was varied. However, it should benoted that if multiple target analytes are present, there is significantpotential for “interferences” (i.e., overlaps between the Ramananti-Stokes spectra of the various target analytes). Depending on theanalytes that comprise each sample mixture, investigation into theactual or potential overlaps must be carried out in order to map outthese issues and mathematically determine the spectra and peaks as afunction of each target analyte concentration or as a function of thepresence of multiple analytes. Additionally, the Raman anti-Stokes peaksused to determine the concentration of the analyte need to be directlycorelated with the concentration. In general, one would require multiplepeaks to generate the correlation or correlation function to theconcentration.

BRIEF SUMMARY OF THE INVENTION

The invention described and claimed herein is an analyzer system fordetermining the identity and concentration of at least one targetanalyte present in a gaseous or liquid sample utilizing the Ramanoptical scattering effect, said analyzer system comprising a Ramananti-Stokes analyzer apparatus that utilizes HCPBG fiber to spatiallyconfine both the pump and signal light to thereby increase theinteraction length and hence the Raman anti-Stokes emission signalmagnitude. The fact that the anti-Stokes light can be efficientlyexamined (as opposed to the Stokes radiation heretofore utilized by theprior art) allows for increased immunity to the effects ofauto-fluorescence. The spontaneous Raman anti-Stokes signal is notreadily used as the signature for analyte quantification due to the factthat the anti-Stokes amplitude is typically significantly (2-3 orders ofmagnitude) lower than the Raman Stokes signal.

Another impediment to accurate, real-time, analyte quantificationalleviated by the present invention is the necessity for training setsand multivariate analysis (e.g., Principal Component Analysis and PLS),though these techniques can, if desired, still be used with theapparatus described herein. However, the necessity for such techniquesis avoided for most analyte samples by several unique and advantageousfeatures of the present invention particularly the use of knowncalibrant sets (compounds corresponding to the target analyte oranalytes, preferably at predetermined concentrations) in the systemwhich can be done without compromising sterility if desired ornecessary. Additionally, the present invention provides the artworkerwith:

-   -   1. The ability to employ univariate analysis based on the        spectra obtained using the apparatus of the present invention        when using calibrant sets of known concentration;    -   2. The ability to monitor the pump light amplitude launched into        the HCPBG fiber and the remaining pump light magnitude (as well        as the Raman Stokes and Raman anti-Stokes emissions) exiting the        fiber thereby allowing for a measure of scattering and/or        absorption loss in the fiber.

These same aforementioned sets of calibrants can also be used tovalidate the operation of the sensor system in a cGMP or medicalenvironment. The present invention is also advantageous in that it doesnot always require the use of a filter to reduce the scattering densitysince it generally avoids the problem of auto-fluorescence, therebyresulting in a cleaner spectrum. It should be noted however that hecalibration system of the present invention can also be advantageouslyutilized in the case of Raman Stokes spectra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 are illustrative of the prior art. FIGS. 6-13 are illustrativeof the present invention.

FIG. 1 shows a prior art sample Raman Stokes spectrum withauto-fluorescence in the graph labeled 1. The trace labeled 2 is theRaman Stokes signal of the fluid with no processing, while trace labeled3 is the Raman Stokes signal post ultrafiltration. The graph 4 shows theRaman Stokes signal with the auto-fluorescence subtracted out using a5th order polynomial fit: The trace labeled 5 is the native (unfiltered)sample and the trace labeled 6 is the signal for the ultrafiltrationsample.

FIG. 2 shows a prior art Raman Stokes spectroscopy system utilizing aTeflon AF hollow core waveguide and a white light source spectrometer tomeasure the absorption and scatter loss in real time; this lossmeasurement is implemented in the concentration calculation based on theRaman Stokes signal. In this system, 20 is an 830 nm laser, 201 is a CDbased spectrograph. 202 is a spectrometer, 203 is a power meter, 204 isa white-light source, 205 is the LCOF, 206 is a dichroic beam splitter,207 is an edge filter, and 208 is a band pas filter.

FIG. 3 shows a prior art Raman anti-Stokes flow cell and free spaceoptical collection system as described in the aforementioned article byDou et al. In this figure, 30 are the sample fluid entrance and exits ofthe optically transparent quartz flow cell 305. The flow cell issurrounded by a gold coated “clam-shell” formed by 306 and 307. The pumplaser enters the flow cell chamber 315 in the clamshell through 310 andthe Raman anti-Stokes emission light is collected after the conicallyshaped exit 320.

FIG. 4 shows both the Raman Stokes and Raman anti-Stokes spectrumcollected using the prior art cell shown in FIG. 3. The spectrum 401shows the Raman Stokes signal while spectrum 402 shows the anti-Stokesspectrum.

FIG. 5 shows a prior art fit and correlation between glucoseconcentration and a Raman anti-Stokes peak at 130 cm⁻¹ with the datacollected using the prior art system shown in FIG. 3.

FIG. 6 shows a commercially available hollow core photonic band gapfiber suitable for use in the present invention.

FIG. 7 shows one embodiment of the present invention.

FIG. 8 depicts a pump scheme and monitoring system suitable for use inthe present invention.

FIG. 9 shows a detection and spectral analysis system suitable for usein the present invention.

FIG. 10 shows an alternative detection system based on spectral peakdetection suitable for use in the present invention.

FIG. 11 depicts an embodiment of the present invention that allows forcalibration and validation of the system.

FIG. 12 illustrates an embodiment of the invention specifically adaptedfor bioprocess monitoring.

FIG. 13 depicts a custom HCPBG fiber having more than one core.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 6 shows a commercially available hollow core photonic band gapfiber suitable for use in the present invention. The hollow core isshown here as 601, while the photonic band gap based cladding is shownas 602 and the solid wall surrounding the fiber is shown as 603.

FIG. 7 shows one embodiment of the present invention where 701 is thepump laser (excitation light) source that is described in further detailin FIG. 8. The fiber transmitting the pump light is shown as 702 while703 is a splice protector covering the splice 704 between 702 and theHCPBG 708. The splice protector is hermetically sealed to the fibers forexample by adhesive 707 which is suitably USP Class VI and gammaradiation resistant to at least 50 kGy as are preferably also the spliceprotector and the fiber. An inlet hole 705 through the splice protectorand an inlet hole 706 through the fiber into the hollow fiber coreallows a fluid sample, generally liquid (although a gaseous sample isalso amenable to analysis in accordance with the present invention), tobe drawn or dispensed by pump 710 through a conduit 709 such that itenters the core of the fiber. We refer to the liquid or gas to be drawninto the analyzer as a sample which can be comprised of either a singleanalyte or plurality of analytes to be identified and its (their)concentration(s) quantified. The hole is preferably (though notabsolutely necessarily) within a few millimeters of the splice so thatthe entire fiber core on both sides of the hole can be readily filedwith fluid thereby creating a homogenous index for optical propagationonce present in the core. The fluid can also be pulled by pump 710through the core after aces from a similar splice protector 717 and acesholes therein. This second aces port on the exit side of the assemblycan be coupled to a tube 71 so that the fluid can be drawn through acentrifuge, filtration, or ultra-filtration system 713 which, ifdesired, can be remotely located from the splice and hole. The exitfiber 714 is connected to the HCPBG fiber identical to that described onthe input side with a splice protector and holes similarly positioned.The light from exit fiber 714 is directed to detection system 715.

The invention described in FIG. 7 allows the amplitude of the Ramananti-Stokes signal to be increased by increasing both the pump power andthe interaction length without suffering the same deleterious effects offluorescence as would be incurred by a Raman Stokes signal. In general,many parasitic signals like fluorescence will appear on the Stokes side(red shifted) of the spectrum but not on the anti-Stokes (blue shifted)side. However, even with an anti-Stokes signal it is generally desirableto minimize scattering loss, but the scattering and absorption loss canbe accounted for to a first order in the present invention. Also, theuse of filtered samples can significantly reduce the presence ofscatters. Any remaining scattering and absorption loss can be accountedfor by noting the pump loss as it travels through the fiber beforeinitial use; the 30 pump loss will be both qualitatively andquantitatively indicative of the loses experienced by the Ramananti-Stokes signal. Cut back experiments (a technique well known in theart in which optical loss in which plural segments of the fiber ismeasured) on the fiber can be used to baseline the attenuation of thepump light in the system when filed with a liquid that does not absorbor scatter in the wavelength regime of interest (e.g., sterile water oraqueous pH buffer 35 solution) and monitoring of the power converted tothe Raman Stokes and Raman anti-Stokes allows for a complete accountingof the input power In FIG. 7, as indicated, the excitation light sourceis shown as 701. For a suitable wavelength range (e.g., approximately350 nm to 850 nm) the fiber core diameters will generally be in therange of 0.60 microns or less. This is further detailed in FIG. 8 whichshows a wavelength stable, narrow line-width laser 81, preferablyemitting in the range of 785 nm to 810 nm. This laser wavelength rangeis preferred in order to maintain a single- or close-to-single modeoperation of a larger diameter (>40 micron) HCPBG fiber. Althoughultraviolet and visible wavelength lasers can be used with the benefitof increased Raman cross-section, a downside is the increased number oftransverse modes (for a fiber of given core diameter) and therefore thepotential for reduced interaction length and noise. An optimal systemwill excite only a fundamental mode in the HCPBG fiber, or a smallnumber of transverse modes (e.g., less than approximately 15) such thatthe loss in the HCPBG fiber is deterministic and is relativelyconsistent (e.g., less than approximately 7% variation) in a multitudeof units built the same way. The mode number can be calculated as perknown procedures (see Experimental demonstration of the frequency shiftof bandgaps in photonic crystal fibers due to refractive index scaling,G. Antonopoulos, F. Benaim, T. A. Birks, D. M. Bird, J. C. Knight, andP. St. J. Russell, Opt. Express 14(7), p. 3000, 2006).

As mentioned above, one embodiment of the invention disclosed andclaimed herein is shown in FIG. 7 where the excitation lighttransmission fiber 702 is attached to the HCPBG fiber by fusion splicingor other suitable means of producing a low los optical connection. Thesplice or connection between the fibers on the entrance and the exitends of the HCPBG fiber is protected from physical damage by cover orsleeve 703. Splice protector 703 also includes a hole through whichfluid can enter and exit the HCPBG fiber. A sample can thus be pulled bya pump or syringe or other suitable dispenser 710 through the HCPBGfiber. A suitable dispenser can be any type of miniature pump as arewell known in the art including printer ink-jet cartridge type devices.If the dispenser and/or pump is to be used bi-directional, then thefluid to be analyzed can either be a calibrant injected by 710 throughconduit 709 or alternatively can be a sample pulled up through theoptional filtration/ultra-filtration system 713. The aces holes areplaced in the HCPBG fiber through the cladding into the core. This holeis optimally produced by laser drilling and preferably by CO₂ or F₂laser drilling using known techniques where the hole is created bythermal ablation thereby also sealing the photonic band gap claddingholes simultaneously. (see Laser drilling and routing in optical fibersand tapered micropipettes using excimer, femtosecond, and CO2 lasers,Armitage et al, Proc. SPIE 5578, 596 (2004)) The core diameter of HCPBGfiber plays a role in determining whether or not it is a single-modewaveguide. In general, the fiber core diameters preferably range fromabout 10 microns to about 100 microns depending on the desired singlemode cutoff wavelength and the photonic band gap cladding design. Aspreviously indicated, for a suitable excitation light wavelength range(e.g., approximately 350 nm to 850 nm) the fiber core diameters willpreferably be in the range of 60 microns or less. It is desirable tohave both the ingress and egress holes approximately the same size asthe core diameter thereby allowing the core to be filled and emptiedwith minimized pressure differential requirements. As mentioned before,the core can be filled by using a manual or automated dispenser 710 topass the sample through a centrifuge or ultra-filtration system 713 orto inject a sample of known concentration of the analytes under study.(see Recent Developments in Membrane Based Separation in BiotechnologyProcesses: Review, A. S. Rathore, A. Shrike, in Preparative Biochemistryand Biotechnology, Vol. 41 (4), 398, 2011).

The advantage of using an active or passive filtering system throughwhich the sample is passed is to reduce the density of scatters and/orabsorbers in the measured sample, and thereby help provide a clean Ramanspectrum such as is shown in FIG. 1. Filtering is not always required,but will generally enhance the clarity of the spectrum. An advantage ofthe present invention is that the use of the anti-Stokes radiationminimizes auto-fluorescence and therefore will substantially clean upthe spectrum even without filtration or centrifugation. All wettedcomponents in FIG. 7 can readily be created from USP Class VI, gamma orBeta radiation stable, and animal component derived free materials.

FIG. 8 depicts a pump (excitation light) source for use in the presentinvention where 81 is the laser source, and the light 82 is coupled intofiber 87 using a lens or other optical system 84. A portion of the beamis reflected using beam splitter 83 and the power (e.g., power) ismeasured using detector or detector/filter combination 85. Beam splitter83 can suitably be positioned either before or after the optical system84 depending on the details of the implementation and beam divergenceand the beam splitter's optical characteristics. In FIG. 8 beam splitter83 allows a known portion of the pump light to impinge upon a detector85 that preferably will have a filter in front of it to ensure thefidelity of the signal. The beam can be collimated or re-focused byoptics 84 and while the optics are shown after the pick off, they canequally be placed before or with the pick-off in between two lenses orsimilar phase changing optical elements. The pump light is subsequentlycoupled into a single mode or relatively low V number (se e.g., OpticalWaveguide Theory, A. W. Snyder, J. Love, Springer, 1983) input fiber,87, that can be fusion spliced or otherwise physically coupled to theHCPBG fiber. After passage through the HCPBG based Raman anti-Stokesanalyzer the Raman Stokes light, the pump light, and the Ramananti-Stokes light are coupled into the exit fiber which is also splicedto the HCPBG as shown in FIG. 7.

FIG. 9 shows a detection system suitable for use in the presentinvention. In FIG. 9 the exit fiber, 910, delivers the Raman Stokes,pump, and Raman anti-Stokes light 920 that is collimated by a lens orother system of optical elements 930 after which a dichroicbeam-splitter is used to separate the pump light or the pump light andthe Raman Stokes light. The separated light impinges upon a filter 940which can let both the Raman Stokes and pump light onto detector 942, oralternatively the filter 940 can be a dichroic reflector which allowseither the Raman Stokes signal or the pump light to pas and reflects theremaining pump or Raman Stokes signal to second detector 94. Since thecutback experiments on the fiber provide the fundamental loss of thefiber, and the excitation system of FIG. 8 allows one to know andmonitor the amplitude of the pump light launched into the fiber, andsince one also knows from the detection system of FIG. 9 the totaltransmitted optical power that is not either the Raman Stokes or theRaman anti-Stokes signal, one can therefore determine the absorption andscattering los of the analyte under test. Specifically, the presentinvention makes it possible to account for all of the pump power that islost in transmission through the HCPBG fiber and that is converted tothe Stokes and Anti-Stokes signals. Therefore, the bulk of the pumploses can be attributed to scatter and/or absorption allowing for thecalculation of a los coefficient. The spectral extent of the RamanStokes and Raman anti-Stokes signals of interest are on the order of 50nm to the red shifted and blue shifted sides of the pump wavelength,respectively, and the scattering and absorption functions will notgenerally change value significantly in this region. In FIG. 9 the exitfiber is shown as 910 and light exiting the HOB fiber couples directlyinto the exit fiber. Light 920 from the fiber is collimated by lens oroptical system 930. Dichroic beam-splitter 950 sends pump and RamanStokes light and remaining pump light to band-pas filter/detector pair35 940/945 where the pump light amplitude is passed and measured and theRaman Stokes light reflected. The Raman Stokes amplitude is measured atdetector 94. The remaining light, i.e., the Raman anti-Stokes signal,impinges on dispersive element (e.g. a ruled or holographic grating) 960and is dispersed to detector array or CD 970. The resultant electricalsignal is carried to a data signal processing unit or other similarcomputer system by cable 980. The entire Raman anti-Stokes spectrum cannow be examined for the spectral signatures of the analytes under study.This allows the spectral features of the analyte's Raman anti-Stokessignal to be fully analyzed. If, as is generally the case inbioprocessing, the basic make-up of the analyte containing media andanalytes are known, it is feasible to create a map of the spectral peaksof interest. This map can be used in conjunction with the data from 980and aforementioned data signal processing system to identify the analyteconcentrations using univariate analysis or peak fitting/area analysis.

FIG. 10 shows a detection system where 101 is the exit fiber and theremaining pump, Raman Stokes, and Raman anti-Stokes light beam 102 fromthe exit fiber is collimated by lenses or optical system 103. The lightbeam hits dichroic beam splitter 104 and the pump and Raman Stokes lightis sent to dichroic beam splitter 105 which causes the pump light to hitfiltered detector 106 and Raman Stokes light to hit band pas opticalfiltered photo detector 107 thus allowing analysis of scattering los inthe system with the pump power as the reference. A dichroicbeam-splitter array is depicted by 108 where Raman anti-Stokes spectralregions of interest transmitted by 104 are split of and sent to adiscrete optically filtered detector array 109. The resulting electricalsignals are carried by a set of cables or wires 10 to a data signalprocessing system such as for example a PC type computer. The dichroicelements are chosen to correspond to the known spectral features of thesample under test. For samples comprised of a small number (<˜15) ofanalytes where the amplitudes of spectral peaks have been found todirectly corelate to concentration have already ben mapped out, thisdetection system is especially simple and cost effective.

FIG. 11 shows an embodiment of the present invention that allows for theuse of multiple calibrants or multiple concentrations of one analyte orany combination thereof such that the system's base-line response can beestablished. Specifically, if it is known that certain analytes will bepresent in the sample it is possible to take into account any crosssensitivities or spectral peak overlaps by having available knownconcentrations of the analyte or admixtures/combinations of multipleknown concentrations of analytes available such that the spectrum can betaken of the system that will be used to quantitatively analyze thesamples under test. For example of it is known a priori that the samplewill mainly consist of water, buffering systems for pH stabilization(e.g., borate or phosphate based solutions), glucose, lactose, urea,glutamine, glutamate, and some specific additives to help cell growth asis commonly found in buffered media during cell growth processes, onecan map out the maximum and minimum concentrations and provide premixed,sterile samples contained in 129 as shown in FIG. 11 such that thesystem response of the analyzer can be automatically pre-calibrated.Specifically, the peaks can be identified, and amplitudes correlated toanalyte concentration and cross-sensitivities can be also be mapped out.This can also be done in a similar fashion for blood, food/beverage,chemistry, and wastewater analysis or any sample where the sampleconstituents are known to a reasonable degree of certainty in advance.Subsequently, samples can be taken in real time and the spectrumanalyzed using univariate analysis to quantitatively and accuratelyyield the concentrations of the analytes that make up the sample. (seeData, models, and statistical analysis, A. Cooper, Tony J. Weekes,Rowman & Littlefield, 1983, ISBN 0389203831). Additionally, analytesamples 129 can also be used in a cGMP setting to allow validation ofthe system. (see GAMP5, A Risk-Based Approach to Compliant GxP,Computerized Systems, 2007, ISPE.) In the present invention, the work ofcreating training sets for use in multi-variate analysis (e.g., PLS orPCA) can be optionally replaced with a more deterministic method ofsystematically creating calibrants. The system described herein can beused with multi-variate analysis as well. FIG. 11 depicts an embodimentof the present invention that allows for calibration and validation ofthe system. The excitation light optics 120 connects to the HCPBGoptical fiber system 122 through the excitation fiber 121 and travelsthrough the exit fiber 123 to the detection system 124. The fluid can bedrawn into or pushed out of the core of HCPBG fiber through inlet 130.Calibration or validation liquids can be introduced into the core of theHCPBG using dispensing units 129 or subsequently withdrawn into emptydispensers. These units, 129, are connected to a common conduit 128 toenter the HCPBG optical fiber system 122.

FIG. 12 depicts an embodiment of the present invention that isparticularly well suited for use in bio-processing applications such asin a bioreactor, a mixer for production of media or buffers or similarproducts, or in a bioprocessing down-stream processing skid. In thisapplication, the very small bend radius (typically <1 cm) of HCPBG fiber12 can be exploited by wrapping it around a small diameter mandrel 11preferably constructed of USP Class VI ultra-low density polyethylene orcomposite material such as is described in co-pending, commonly assignedU.S. patent application 13/385,10. The HCPBG fiber 12 is wrapped arounda solid disk and mandrel 11 that can be attached to the wall of apolymeric bioreactor vessel 19. The pump entrance and exit optics 18 isattached to the input fiber 14 and the detection system 18 is connectedby the exit fiber 15. The fluid sample enters and exits the HCPBG fiberthrough ports 16. If desired the fluid can be pulled into the core ofthe HCPBG fiber through a filtration or ultra-filtration system 17 whichis attached to excitation fiber 14. The fluid introduction path wherethe splice and splice protection occurs (between 14 and 12) is shownhere as 13. The exit fiber is shown as 15 connected to detection system18. The bioreactor, mixer, or other system is shown here as having aflexible polymer wall 19, alternatively this can also be a wall thatcomprises part of a plastic vessel that this embodiment of the inventionis bonded to. In general practice, the inner layer of this wall in asingle-use bioreactor or mixer is comprised of ultra-low densitypolyethylene or similar high surface energy polymer (>20 dynes/cm.) andcan be fusibly sealed to the base of mandrel 11. Fluid port 16 on theexit side is shown leading to the inside of the container and isterminated by a filtration or ultra-filtration system 17. A similarsystem can be created that sits outside the system (e.g.: bioreactor,mixer, downstream processing system) where the fluid sample input port17 leads to a pump or sampling system followed by a centrifuge, or otherfiltration system that has already taken a sample and processed it toremove scatterers thereby leaving the fluid with a quality similar tothat of a supernatant liquid in that the chemical composition iscomplete, but the precipitant or other solids have been removed. Withthe use of ultra-filtration and minimized scattering of the pump andsignal beams, the clarity of the spectrum approaches that seen byMeneghini et al in the Raman Stokes spectrum of ethanol where theeffects of scatterers and auto-fluorescence were both minimal.Therefore, the quantitative analysis of the spectrum becomes similarlystraightforward and the need to keep track of the pump loses iseliminated.

FIG. 13 depicts a custom HCPBG fiber having at least two cores. Forsimple systems of analytes, it is possible as shown in FIG. 13, tocreate HCPBG fibers with multiple hollow cores. Some of these cores canbe pre-filed with calibrants fluids of known and certified/validatedanalyte concentration such that the system is pre-configured and readyto use. The only operational change in the system is the addition of theability to switch the entrance fiber and exit fiber light to and fromthe particular fiber of interest respectively. This switching technologyhas existed commercially since the telecom “boom” of the 1990s and nowis commercially available in many formats.

Another embodiment of the present invention immediately applicable tobiotech/bioprocessing utilizes two or more HCPBG fiber channels inparallel along a mandrel thereby enabling one to switch the opticalsignal output with a coupler from sample to reference calibrant inreal-time, thereby eliminating the need to run the sample and calibrantthrough the same physical path, and mitigating customer concerns aboutflushing any of the calibrant through the filter into the bioreactor.

1.-20. (canceled)
 21. An analyzer system for determining the identityand concentration of at least one target analyte present in a gaseous orliquid sample utilizing the Raman optical scattering effect, theanalyzer system comprising: (i) a laser light source emitting lightwhich generates Raman Stokes and anti-Stokes emissions when incident onthe target analyte; (ii) one or more hollow core photonic band-gap(HCPBG) fibers optically connected to the laser light source, HCPBGfibers including: (a) a first inlet permitting introduction of a samplecontaining the target analyte into the HCPBG fibers; and (b) at leastone reference calibrant in the HCPBG fibers, the reference calibrantcorresponding to an analyte in the sample; (iii) a pump configured toinject the sample containing the target analyte into the core of theHCPBG fiber; and (iv) a spectral analysis system optically coupled tothe HCPBG fibers and configured to derive the Raman anti-Stokes spectralpeaks and/or spectra of the reference calibrant to establish a baselineresponse and account for cross sensitivities or spectral peak overlapsin the sample.
 22. The system of claim 21, where the one or more HCPBGfibers comprise a single HCPBG fiber that has multiple hollow channels,wherein at least one of the hollow channels is pre-filled with thereference calibrant.
 23. The system of claim 21, where the one or moreHCPBG fibers comprise two or more HCPBG fibers wound in parallel along amandrel, where at least one of the HCPBG fibers comprises the referencecalibrant, the system further comprising a coupler which switches thelaser light source output from one fiber to the other.
 24. The system ofclaim 21, where the one or more HCPBG fibers comprise a single HCPBGfiber having two or more parallel channels wound along a mandrel, whereat least one of the channels comprises the reference calibrant, thesystem further comprising a coupler which switches the laser lightsource output from one fiber channel to the other.
 25. The system ofclaim 21, where the spectral analysis system comprises at least oneoptically filtered photo-detector and/or optically filteredphoto-detector array.
 26. The system of claim 21, further comprising asample filtration system through which the sample passes before beingintroduced into the one or more HCPBG fibers.
 27. The system of claim21, wherein the sample contains a plurality of analytes having knownconcentrations.
 28. The system of claim 21, further comprising a monitorwhich measures an amplitude of the laser light source before it isintroduced into the one or more HCPBG fibers.
 29. The system of claim21, wherein the spectral analysis system further comprises a monitorwhich measures a magnitude of the laser light source, the Raman Stokesemission signals, and the Raman anti-Stokes emission signals after exitfrom the one or more HCPBG fibers and thereby determines the scatteringand/or absorption loss.
 30. The system of claim 21, further comprisingmeans for determining the concentration of the target analyte or otheranalytes in the sample using multi-variate analysis of the Ramananti-Stokes spectra and corresponding training sets.
 31. The system ofclaim 21, further comprising means for determining the concentration ofthe target analyte or other analytes in the sample using univariateanalysis of the Raman anti-Stokes spectra and the correspondingcalibrant spectra.
 32. The system of claim 21, wherein the laser lightsource emits light in the range of 350 to 850 nm.
 33. A system for thedetermination of the concentration of at least one target analytepresent in a gaseous or liquid sample using Raman anti-Stokes radiationspectroscopy the system comprising: (a) a laser pump light source; (b)an inlet for the laser pump light source into one or more hollow corephotonic band gap (HCPBG) fibers, the HCPBG fibers operating in afundamental mode or a limited number of modes, the HCPBG fibersconfigured to contain a reference calibrant; (c) a pump configured tointroduce the sample containing the at least one target analyte into thecore of the HCPBG fiber via an inlet port; (d) a spectral analysissystem comprising at least one filter and/or dispersive element and adetector array or CCD including means for detecting and quantifyingRaman anti-Stokes spectrum and/or spectral peaks emitted by thereference calibrant to establish a baseline response and account forcross sensitivities or spectral peak overlaps in the sample whenilluminated by the laser pump light source; (e) means for opticallycoupling components (a) and (d) to the core of the HCPBG fiber.
 34. Thesystem of claim 33, where the laser pump light source includes anamplitude monitor.
 35. The system of claim 33, further comprising meansfor monitoring the amplitudes of the signals that exit the hollow corephotonic bandgap fiber and account for scattering and/or absorption lossof the Raman anti-Stokes signal.
 36. A method of determining theidentity and concentration of at least one target analyte present in agaseous or liquid sample utilizing the Raman optical scattering effect,the method comprising: exciting a reference calibrant in one or morehollow core photonic band-gap (HCPBG) fibers, using an excitation lightsource, the reference calibrant corresponding to a target analyte in asample; as a result of exciting the at least one reference calibrant,deriving the Raman anti-Stokes spectral peaks and/or spectra of thereference calibrant to establish a baseline response, using a spectralanalysis system optically coupled to the HCPBG fibers; introducing thesample containing the target analyte into the one or more HCPBG fibersthough an inlet in fluid communication with a hollow core of the HCPBGfibers; exciting the sample using the excitation light source; as aresult of exciting the sample containing the target analyte, derivingthe Raman anti-Stokes spectral peaks and/or spectra of the targetanalyte from the Raman anti-Stokes light received and accounting forcross sensitivities or spectral peak overlaps in the sample containingthe target analyte; and expelling the sample containing the targetanalyte from the hollow core of the HCPBG fibers.
 37. The method ofclaim 36, further comprising determining a concentration of the targetanalyte in the sample using multi-variate analysis of the Ramananti-Stokes spectra and corresponding training sets.
 38. The method ofclaim 36, further comprising switching the excitation light source froma fiber having the reference calibrant to a different fiber having thesample.
 39. The method of claim 36, further comprising measuring anamplitude of the excitation light source before it is introduced intothe one or more HCPBG fibers.
 40. The method of claim 36, furthercomprising measuring the magnitude of signal from the excitation lightsource, the Raman Stokes emission signals, and the Raman anti-Stokesemission signals after exit from the one or more HCPBG fibers andthereby determining scattering and/or absorption loss.